Membrane and method for culture and differentiation of cells

ABSTRACT

Provided is a membrane for cell culture and differentiation. The membrane has a base portion and an array of protrusions consisting of a plurality of protrusions. The protrusions are substantially evenly distributed on the base portion. The plurality of protrusions has dimensions on the order of micrometers. In particular, the membrane consists of particles of different particle sizes of two or more kinds. One kind of particles have an average particle size of 1 μm to 50 μm. Two or more kinds of particles of different particle sizes include nanoscale particles, 10-900 nm. One kind of particles are selected from the group consisting of inorganic compound microspheres. The other kind of particles of the two or more kinds of particles of different particle sizes are selected from the group consisting of organic polymer nanospheres. Also provided is a method for maintaining, culturing and/or differentiating cells using such membrane.

TECHNICAL FIELD

The present disclosure relates to the technical field of cell cultureand cell differentiation. In particular, the present disclosure relatesto the field of cell differentiation using a multi-scale particlemembrane. More particularly, the present disclosure relates to aone-step method for expansion of human induced pluripotent stem cells(hiPSCs) and directed differentiation of hiPSCs into maturecardiomyocytes using the multi-scale particle composited membrane.

BACKGROUND ART

In recent years, stem cell-based research has made rapid progress,making it possible to cure heart-related diseases. Human inducedpluripotent stem cells (iPSCs) belong to pluripotent stem cells that candifferentiate into functional cardiomyocytes in vitro, which may be usedfor repairing a heart. Since iPSCs can be derived from autologous cells,there are no issues such as ethics and immunosuppression. Since directtransplantation of iPSCs has a high risk of tumorigenesis, it is abetter treatment means to first differentiate iPSCs into cardiomyocytesin vitro before transplantation. iPSC-CMs are cardiomyocytes (CMs)obtained by inducing directed differentiation of iPSCs, and are the bestcell type for construction of disease models in vitro, drug screeningand cell transplantation treatment. Studies have shown that in mouse andporcine myocardial infarction models, iPSC-CMs can survive in a heart ofa host and improve cardiac functions after transplantation. hiPSC-CMsexhibit many characteristics that are the same as the characteristics ofnormal cardiomyocytes in vivo, such as morphological structure, geneexpression, functional ion channels, receptor expression, andelectrophysiological properties. These characteristics make hiPSC-CMs agood model for drug cardiotoxicity testing in vitro. At present, theobstacle to improving the efficiency of new drug research anddevelopment is that in vitro cardiomyocytes are not mature enough, whichmakes it impossible to accurately predict drug toxicity. High yield andhigh maturity are the prerequisites and key for the reliable applicationof hiPSC-CMs in drug screening in vitro.

Current methods for inducing differentiation of hiPSCs into hiPSC-CMsinclude (1) 3D induction method based on embryoid bodies (EBs) and (2)2D induction method of single cell seeding.

The 3D induction method based on EBs is to first culture the hiPSCsuspension to form EBs, and then inoculate the EBs on feeder cells orextracellular matrix (for example, Matrigel) for adherent culture, whichfinally obtains, by virtue of the interaction between cells in the EBsand the factors in the culture medium, the hiPSC-CMs with spontaneouscontraction and rhythmic beats. This method involves two steps and haslow differentiation efficiency, and in this method cells within the EBsare highly different. For these shortcomings, the 2D induction method ofsingle cell has recently been widely utilized.

In the 2D induction method, cells are directly contacted with solublebiochemical factors in a medium to cause rapid differentiation of thecells. Initially, in the 2D induction method, mouse visceralendoderm-like cells (END-2) were used as feeder cells to produceActivin-A and BMP factors to promote the formation of ventricular-likecardiomyocytes. Recently, the addition of biochemical factors orchemical small molecules has increased controllability of intracellularsignaling pathways, which has become a main means of myocardialdifferentiation. A “matrix sandwich” method using Matrigel combined withgrowth factors Activin-A/BMP4/FGF2 can also increase cardiomyocyteproduction. In clinical use, the above conventional method is stillsubject to certain restrictions, involving, for example, cells andproducts derived from animals, heterogeneity of hiPSC-CMs (incompletelydifferentiated or undifferentiated cells are present in the cellpopulation), and relatively low degree of maturation of cells in thecase of adherent culture on a traditional substrate in a 2D manner.Thus, currently it is still a main problem to get high-purity andhigh-maturity hiPSC-CMs.

In summary, the prior art for stimulating the differentiation of hiPSCsinto hiPSC-CMs has the following disadvantages.

1) In the traditional 3D method based on EBs, it is necessary to firstprepare EBs, and then transfer EBs from suspension culture to adherentculture, which is time consuming and inefficient.

2) The traditional 2D method is to culture hiPSCs using atwo-dimensional planar surface and obtain hiPSC-CMs by adding smallmolecule compounds or growth factors, which may result in low cellmaturity due to failure to meet bionic requirements.

3) Since layered iPSC-CMs are obtained by the traditional 2D method, adigestive enzyme should be used to remove the cells from the surface inthe later stage, which may cause damage and quantity loss to the cells.

SUMMARY OF THE INVENTION

The present disclosure provides a membrane for culture anddifferentiation of cells, comprising:

(A) a base portion; and

(B) a protrusion array composed of a plurality of protrusionssubstantially distributed on the base portion, the plurality ofprotrusions having a size on the order of micrometers.

The present disclosure also provides a method of culture and/ordifferentiation of cells, comprising culturing and/or differentiatingthe cells on the membrane of the present disclosure.

The present disclosure also provides a method for maintaining growth andstemness of cells, comprising culturing cells on the membrane of thepresent disclosure, wherein the cells are selected from the groupconsisting of induced pluripotent stem cells, embryonic stem cells, oradult stem cells.

BRIEF DESCRIPTION OF DRAWINGS

In order to more clearly illustrate embodiments of the presentdisclosure or technical solutions in the prior art, drawings to be usedin the description of embodiments or the prior art are briefly describedbelow.

FIG. 1 generally illustrates a process of an embodiment of the presentdisclosure, including a one-step process of inducing directeddifferentiation of hiPSCs into hiPSC-CMs on a multi-scale particlemembrane, and functional identification of the resultant hiPSC-CMs.

FIG. 2 shows a schematic diagram of a precipitation-based self-assemblyof a single layer binary colloidal crystal (BCC) membrane as an exampleof a multi-scale particle membrane, wherein A represents large-sizedparticles, B represents small-sized particles, and C represents anO-ring.

Panels 2PS, 5PS, 2PM and 5PM in FIG. 3 respectively represent thesurface SEM images of four binary colloidal crystal (BCC) membranes 2PS,5PS, 2PM and 5PM as examples of multi-scale particle membranes, wherein2PS is composed of 2 μm silicon dioxide (SiO₂) microspheres and 0.2 μmpolystyrene (PS) nanoparticles, 5PS is composed of 5 μm SiO₂microspheres and 0.4 μm PS nanoparticles, 2PM is composed of 2 μm silicamicrospheres and 0.1 μm poly(methyl methacrylate) (PMMA) nanoparticles,and 5PM is composed of 5 μm SiO₂ microspheres and 0.2 μm PMMAnanoparticles. In FIG. 3, A represents larger particles, and Brepresents smaller particles distributed between the larger particles.

FIG. 4 shows a flow chart of culture and differentiation of hiPSCs. Asshown in FIG. 4, four days before the start of cell differentiation,hiPSCs were expanded on Matrigel-coated 4 BCC surfaces and coverslipsurfaces (as control), and then maintained on a mTeSR medium.

When hiPSCs reached 80% confluence, a cell differentiation step isstarted, and the medium was changed to RPMI1640 (Gibco, 1744361) withB-27 (Gibco, A1895601). The cells were also exposed to the GSK313inhibitor CHIR 99021 (6 μM, Selleck, S2924) at the beginning ofdifferentiation, followed by a Wnt antagonist IWR-1 (5 μM,Sigma-Aldrich, 10161). Contracting cells were noted from day 8 and werefed every alternate day with RPMI1640 supplemented with the B-27supplement (Gibco, 17504-044). During day 15-20, the medium was changedto a purification medium, which consists of a glucose-free Dulbecco'smodified Eagle's medium (Gibco, 11966025) supplemented with 4 mM lacticacid and sterile 1M Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES). After 30 days of in vitro differentiation, the cells weretrypsinized and replated on gelatin-coated coverslips (Solarbio, YA0352)for further experiments.

FIG. 5A is a schematic diagram showing morphology of the hiPSCs observedunder a microscope on day 2 from the start of differentiation ondifferent surfaces (scale bar 500 μm), and a comparison of a 2D-likecell population grown on the smooth control substrate with the 3D cellspheroids grown on the BCCs.

FIG. 5B shows the expression of three stemness-related genes OCT4, NANOGand SOX2 in hiPSCs grown on 2PS, 5PS, 2PM and 5PM of the presentdisclosure, relative to those in hiPSCs grown on control substrates, onday 2 from the start of differentiation.

FIG. 6A shows the morphology of the hiPSCs (scale bar 500 μm) observedunder a microscope on day 8 from the start of the differentiation.

FIG. 6B shows cell type analysis (flow cytometry) of MLC2v and MLC2a.The cells show two kinds of fluorescence, MLC2v and MLC2a.

FIG. 6C shows ratios of MLC2v⁺ cells and ratios of MLC2v⁺/MLC2a⁻ cells(a mature ventricular cardiomyocyte subtype) (n=3), in mean±SD.**p<0.005, ***p<0.001, estimated by one-way ANOVA analysis, followed byDunnet test.

FIG. 7A shows α-actinin, α-tubulin and DAPI staining of a singlehiPSC-CM on day 30 from the start of the differentiation (scale bar=50μm).

FIG. 7B shows aspect ratios of hiPSC-CMs (n=40-44, median (interquartilerange), estimated by Nemenyi test).

FIG. 7C shows circularity of hiPSC-CMs (n=40-44, estimated by one-wayANOVA analysis followed by Dunnet test).

FIG. 7D shows the length of the sarcomere (n=40-44, estimated by one-wayANOVA analysis followed by Dunnet test).

FIG. 8 shows expressions of essential cardiac genes ACTC1, TNNT2, RYR2,SERCA2a, SCN5a, KCNJ2, CACNA1c, ITGB1, GJA1, MYH6 and MYH7 on day 30from the start of the differentiation.

FIG. 9A shows the action potential (AP) of hiPSC-CMs on day 30 from thestart of the differentiation.

FIGS. 9B and 9C show AP duration at 50 and 90% repolarization, APD50 andAPD90, respectively.

FIG. 9D shows a ratio of APD50/APD90 (n=10).

FIG. 10A shows staining of N-cadherin, E-cadherin, and DAPI fromhiPSC-CMs on five surfaces on day 30 from the start of thedifferentiation.

FIGS. 10B and 10C show Western blot analysis of E-cadherin andN-cadherin of the five groups (n=3, estimated by one-way ANOVA analysisfollowed by Dunnet test).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave their common meaning as understood by one of ordinary skill in theart to which this invention is related.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such can vary. As used in this specification andthe appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the context clearly dictates otherwise. Theterms “a” (or “an”), as well as the terms “one or more,” and “at leastone” can be used interchangeably herein.

It is understood that wherever embodiments are described herein withlanguage “comprising”, otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

As used herein, the term “multi-scale particle membrane” or “membrane”,when used in culture, growth or differentiation of cells, refers to asubstrate for culture, growth or differentiation of cells. The term“multi-scale particle membrane” or “membrane” includes two-scaleparticle membrane, three-scale particle membrane, four-scale particlemembrane, and the like. The term “multi-scale particle membrane” or“membrane” includes a monovalent particle membrane, a binary particlemembrane, a ternary particle membrane, a quaternary particle membrane,and the like. The “multi-scale particle membrane” or “membrane” ispreferably a BCC membrane with different particle sizes.

The term “colloidal crystal” is a two-dimensional or three-dimensionalordered array structure that is formed by assembly of monodispersemicrometer-, submicrometer- or nanometer-sized inorganic or organicparticles (also called colloidal particles) via gravity, electrostaticforce or capillary force, similar to a crystal with an atom or moleculeas a standard repeating unit. One example of a colloidal crystal in thedisclosure is a colloidal crystal composed of micrometer-sized silicaand nanometer-sized poly(methyl methacrylate).

The term “cell” is herein used in its broadest sense in the art andrefers to a living body which is a structural unit of tissue of amulticellular organism, is surrounded by a membrane structure whichisolates it from the outside, has the capability of self-renew, and hasgenetic information and a mechanism for expressing it. Cells used hereinmay be naturally-occurring cells or artificially modified cells (e.g.,fusion cells, genetically modified cells, etc.).

As used herein, the term “stem cell” refers to a cell being capable ofself-renew and having pluripotency. Typically, stem cells can regeneratean injured tissue. Stem cells herein may be, but are not limited to,embryonic stem (ES) cells or tissue stem cells (also calledtissue-specific stem cell, or somatic stem cell). Any artificiallyproduced cell which can have the above-described abilities may be a stemcell.

“Embryonic stem (ES) cells” used herein are pluripotency stem cellsderived from early embryos. An ES cell was first established in 1981,which has also been applied to production of knockout mice since 1989.In 1998, a human ES cell was established, which is currently becomingavailable for regenerative medicine.

The term “pluripotency” as used herein refers to the ability of a cellto differentiate into cells derived from any of the three germ layers:endoderm (e.g., interior stomach lining, gastrointestinal tract, thelungs), mesoderm (e.g., muscle, bone, blood, urogenital system), orectoderm (e.g., epidermal tissues and nervous system). “Pluripotent stemcells” used herein refer to cells that can differentiate into cellsderived from any of the three germ layers.

The term “induced pluripotent stem cells” or “iPSCs” refers to a type ofpluripotent stem cells artificially prepared from non-pluripotent cells.Cardiomyocytes derived from iPSCs are a useful experimental system whichhas great potential. They offer an innovative human preparation forcardiac repair, drug safety design and testing, clinical diagnosis, andresearch. Cardiomyocytes derived from iPSCs offer the opportunity towork on cells which recapitulate the activity of healthy humancardiomyocytes, which are otherwise rarely available for comprehensiveexperimental investigation. Human iPSC (hiPSC)-derived cardiomyocytesoffer the ability to develop predictive tools for cardiac function.

As used herein, “differentiation” refers to a change that occurs incells to cause those cells to assume certain specialized functions andto lose the ability to change into certain other specialized functionalunits. Cells capable of differentiation may be any of totipotent,pluripotent or unipotent cells. For mature adult cells, differentiationmay be partial or complete.

“Differentiated cell” refers to a non-embryonic cell that is present ina particular differentiated, i.e., non-embryonic, state. The threeearliest differentiated cell types are endoderm, mesoderm, and ectoderm.The hiPSC-CM as described herein is a cardiomyocyte obtained by inducingdifferentiation of pluripotent stem cells.

The present disclosure provides a membrane for culture anddifferentiation of cells, comprising:

(A) a base portion; and

(B) a protrusion array composed of a plurality of protrusionssubstantially uniformly distributed on the base portion, the pluralityof protrusions having a size on the order of micrometers.

In one or more embodiments, a distance between adjacent protrusions ison the order of micrometers. The distance between the adjacentprotrusions is a distance between the centers of the adjacentprotrusions. In particular, when the protrusions are particles ormicrospheres, the distance between adjacent protrusions is a distancebetween the centers of adjacent particles or a distance between thecenters of adjacent microspheres.

In one or more embodiments, the base portion and/or the plurality ofprotrusions are made of a biocompatible material.

In one or more embodiments, the membrane is a colloidal crystalmembrane.

In one or more embodiments, the protrusions are formed by particles.

In one or more embodiments, the membrane is composed of particles ofdifferent particle sizes of two or more kinds, wherein the two or morekinds of particles of different particle sizes comprise at least:

a first kind of particles serving as the protrusions and having anaverage particle size of from 1 μm to 50 μm, and

a second kind of particles serving as the base portion and having anaverage particle size less than or equal to ½ of that of the first kindof particles.

In one or more embodiments, the two or more kinds of particles ofdifferent particle sizes comprise a first kind of particles serving asthe protrusions and having an average particle size of 1 μm to 10 μm;and a second kind of particles serving as the base portion and having anaverage particle size less than or equal to ½ of that of the first kindof particles.

In one or more embodiments, the protrusions or the particles of thefirst kind have an average particle size of 1-40 μm, 1-30 μm, 1-20 μm,1-10 μm, 1-9 μm, 1-8 μm, 1-7 μm, 1-6 μm, 1-5 μm, 1.5-5 μm, 2-5 μm or2.5-4 μm. The particles of the first kind have an average particle sizeof 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm or 11 μm.

In one or more embodiments, the particles of the second kind have anaverage particle size less than or equal to ½, ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9,1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21,1/22, 1/23, 1/24, 1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/40, 1/50, 1/60,1/70, 1/80, 1/90, 1/100, 1/150, 1/200, 1/250, 1/300, 1/350, 1/400, 1/450or 1/500 of the average particle size of the first kind of particles.

In one or more embodiments, the particles of the second kind have anaverage particle size of 10-900 nm, 20-800 nm, 30-700 nm, 40-750 nm,50-700 nm, 60-650 nm, 70-600 nm, 80-500 nm, 95-400 nm, 100-400 nm,120-380 nm, 140-360 nm, 160-340 nm, 180-320 nm, 200-300 nm or 220-280nm. In one or more embodiments, the particles of the second kind have anaverage particle size of 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380nm, 390 nm, 400 nm, 450 nm, 500 nm, or 600 nm.

In one or more embodiments, the membrane is composed of particles ofdifferent particle sizes of two kinds, wherein in the particles ofdifferent particle sizes of the two kinds, particles of a first kindhave an average particle size of 1 μm to 50 μm, and particles of asecond kind are nano-sized or have an average particle size of 10 nm to900 nm. The protrusions or the particles of the first kind have anaverage particle size of 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-9 μm, 1-8μm, 1-7 μm, 1-6 μm, 1-5 μm, 1.5-5 μm, 2-5 μm or 2.5-4 μm. Theprotrusions or the particles of the first kind have an average particlesize of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm or11 μm. The particles of the second kind have an average particle size of10-900 nm, 20-800 nm, 30-700 nm, 40-750 nm, 50-700 nm, 60-650 nm, 70-600nm, 80-500 nm, 95-400 nm, 100-400 nm, 120-380 nm, 140-360 nm, 160-340nm, 180-320 nm, 200-300 nm or 220-280 nm. The particles of the secondkind have an average particle size of 50 nm, 60 nm, 70 nm, 80 nm, 90 nm,100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm,190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm,280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm,370 nm, 380 nm, 390 nm, 400 nm, 450 nm, 500 nm, or 600 nm.

Without being limited by theory, it is believed that a membrane composedof particles with different sizes of two or more kinds, when serving asa substrate, is advantageous to stemness maintenance and differentiationof cells thereon, producing different effects depending on theproportions and combinations. Since two or more kinds of particles canhave different effects on cell adhesion, for example, some cells preferto adhere to small particles rather than large particles, it is madepossible to regulate the adhesion degree of cells to the surface of theparticle membrane, so as to promote cell-cell interactions, facilitatingthe cells' forming 3D-like spheroids thereon. Such a 3D-like morphologythus facilitates the maintenance of stemness and pluripotency of stemcells in a culture medium. In addition, the formation of such 3D-likespheroid of cells on the membrane of the present disclosure also allowsthe culture, differentiation and harvest of cells to be performed in onestep on the membrane of the present disclosure, without any necessity toperform the steps of first forming EBs and then transferring cells, orthe step of using digestive enzymes after two-dimensionaldifferentiation. Meanwhile, the membrane of the present disclosure alsoallows differentiated cells formed thereon to have higher maturity thandifferentiated cells obtained on a conventional substrate, which isembodied in the function of cardiomyocytes.

In one or more embodiments, the protrusions or the particles of thefirst kind and/or the particles of the second kind are monodisperseparticles.

In one or more embodiments, the protrusions or the particles of thefirst kind are made of an inorganic compound.

In one or more embodiments, the array of protrusions is made of a singlematerial, such as SiO₂, or of multiple materials, such as a combinationof TiO₂ and SiO₂.

In one or more embodiments, the particles of the second kind having adiameter of 10-900 nm are distributed in the array of protrusions. Theparticles of the second kind may be made of a single material, such aspolystyrene (PS) or a mixture of different materials, such as a mixtureof PS and PMMA particles.

In one or more embodiments, the base portion or the particles of thesecond kind are made of an organic polymer, preferably a polymericnanosphere.

In one or more embodiments, the protrusions or the particles of thefirst kind are made of one or more materials selected from the groupconsisting of silica, titania, zinc oxide, chemically modified silica,chemically modified titanium dioxide, chemically modified zinc oxide,and any combination thereof.

In one or more embodiments, the base portion or the particles of thesecond kind is/are made of one or more materials selected from the groupconsisting of polystyrene, acrylic polymer, chitosan,poly(lactic-co-glycolic acid) (PLGA), polylactic acid, polycaprolactone,gelatin and any combination thereof.

In one or more embodiments, the acrylic polymer is selected from thegroup consisting of poly(meth)acrylic acid, poly(meth)acrylate, and anycombination thereof.

In one or more embodiments, the poly(meth)acrylate is selected from thegroup consisting of poly(methyl)acrylic acid C₁-C₂₀ alkyl esters.

In one or more embodiments, the poly(meth) acrylate is selected from thegroup consisting of poly(methyl acrylate), poly(methyl methacrylate),poly(ethyl acrylate), poly(ethyl methacrylate), poly(propyl acrylate),poly(propyl methacrylate), poly(butyl acrylate), poly(butylmethacrylate), poly(pentyl acrylate), poly(pentyl methacrylate),poly(hexyl acrylate), poly(hexyl methacrylate) and any combinationthereof.

In one or more embodiments, the protrusions or the particles of thefirst kind are made of silica, preferably silica microspheres, and thebase portion or the particles of the second kind are made of poly(methylmethacrylate), preferably poly(methyl methacrylate) nanospheres.

Without being limited by theory, it is believed that the combination ofinorganic compound microspheres and organic polymer nanospheres maychemically improve the interaction between the cells and membranesurface, further facilitating the formation of 3D-like spheroids on themembrane of the present disclosure. Therefore, a suitable combination ofinorganic compound microspheres and organic polymer nanospheresfacilitates maintenance of cell stemness of the stem cells, increasesefficiency of cell differentiation, and improves the maturity ofdifferentiated cells. The use of an organic polymer as the particles ofthe second kind or relatively small particles in certain embodiments isalso advantageous in that it can be thermally dissolved or partiallydissolved with an organic solvent, so as to fix the membrane. In someembodiments, the inorganic particles serving as the particles of thefirst kind (protrusions) or relatively large particles is alsoadvantageous in that they have high density and thus are easier toprecipitate, facilitating the preparation of the membrane.

In one or more embodiments, the particles of the first kind are presentas a single layer of particles, and the ratio of the particles of thefirst kind to the particles of the second kind is determined such thatthe particles of the first kind are distributed in the particles of thesecond kind in a partially embedded manner. For example, in the casewhere the particles of the first kind or relatively large particles inthe membrane are arranged in a single layer, 1 cm² area requires about 2μl of a 2 μm particle suspension (10%), or 5 μL of a 5 μm particlesuspension (10%). In some embodiments, the amount of relatively smallparticles or the particles of the second kind serving as fillermaterials is determined in such a calculation manner that a height ofhalf of the diameter of the relatively large particles is reached. Forexample, 1.5 μl of a suspension of second kind of 400 nm particles orrelatively small particles is used for a membrane of first kind of 5 μmparticles or relatively large particles. Because there will be losses inthe process, the above amount is only an estimated value, and the dosein actual production may be higher.

The present disclosure also provides a method for culture and/ordifferentiation of cells, comprising culturing and/or differentiatingthe cells on the membrane of the present disclosure.

In one or more embodiments, the method of culturing cells comprises:

(1) inoculating the cells onto the membrane of the present disclosure,and

(2) culturing the cells.

In one or more embodiments, the method of differentiating the cellscomprises:

(1) implanting the cultured cells onto the membrane of the presentdisclosure, and

(3) inducing differentiation of the cells.

In one or more embodiments, the method for culturing and/ordifferentiating cells comprises:

(1) inoculating the cells onto the membrane of the present disclosure,

(2) culturing the cells in a first medium, and

(3) inducing differentiation of the cells in a second medium,

wherein the step of culturing the cells and the step of inducingdifferentiation of the cells are both performed on the membrane.

The steps of culture and differentiation of the cells in the methods ofthe present disclosure are both performed on the membrane of the presentdisclosure, that is, the method of the present disclosure does notrequire the cells to form into EBs first, for example, by suspensionculture, and then transferred to a culture vessel or scaffold foradherent culture so as to achieve differentiation. Therefore, the methodof the present disclosure is time and effort saving, and also avoidscell damage or quantity loss caused by the transfer process, achievingefficient cell culture and differentiation in one step.

Moreover, the differentiated cells obtained by the method of the presentdisclosure have a higher degree of maturity than those obtained byconventional methods.

In one or more embodiments, the cells are selected from the groupconsisting of induced pluripotent stem cells, embryonic stem cells, oradult stem cells.

In one or more embodiments, the cells are human induced pluripotent stemcells.

In one or more embodiments, the cells are selected from the groupconsisting of bone marrow mesenchymal stem cells, hematopoietic stemcells, neural stem cells, peripheral blood stem cells, adipose stemcells, placental stem cells, placental sub-totipotent stem cells, andamniotic stem cells.

In one or more embodiments, the first medium is selected from the groupconsisting of DMEM, DMEM/F12, RPMI-1640, mTeSR mediums, and anycombination thereof.

In one or more embodiments, the second medium is selected from the groupconsisting of DMEM medium supplemented with B-27, DMEM/F12 mediumsupplemented with B-27, RPMI-1640 medium supplemented with B-27, mTeSRmedium supplemented with B-27 and any combination thereof.

In one or more embodiments, the step of inducing differentiation of thecells further comprises exposing the cells to a GSK3-β inhibitor and/orto a Wnt antagonist.

In one or more embodiments, the cells are induced pluripotent stemcells. By the method of the present disclosure, the induced pluripotentstem cells are differentiated in a directed differentiation manner intohuman induced pluripotent stem cell-derived cardiomyocytes.

In one or more embodiments, the membrane is pre-coated with Matrigel.

In one or more embodiments, the method further comprises separating thecells from the membrane by flushing or suction after the step ofinducing differentiation of the cells. For example, about 8 days afterthe start of directed myocardial differentiation, after formingspherical aggregates, the cells can be separated from the membrane bysuction or flushing.

In one or more embodiments, the flushing is performed with a buffer or aculture medium.

The prior art 2D cell culture and differentiation method requires theuse of an enzyme (e.g., trypsase) for digestion after the completion ofthe culture and differentiation in order to separate the cells from asubstrate, which, however, will cause damages to the cells andcomplicate the culture and differentiation process. For the method ofthe present disclosure, the cells obtained by culture anddifferentiation can be separated from the membrane by simple means, suchas flushing and suction, thus avoiding cell damage and loss in quantity,thereby maximally protecting the cells and simplifying the process.

In one or more embodiments, the membrane is prepared by the followingsteps,

(a) providing a dispersion of the particles of the first kind in a firstdispersion medium,

(b) providing a dispersion of the particles of the second kind in asecond dispersion medium,

(c) distributing the two dispersions on a base material, and

(d) removing the first dispersion medium and the second dispersionmedium such that the particles of the first kind are distributed on thebase material in a single layer and partially embedded in the particlesof the second kind.

The membrane of the present disclosure is simple in preparation, and aplurality of membranes of the present disclosure can be prepared in ashort time, thereby simplifying the process of cell culture anddifferentiation using the membrane of the present disclosure.

In one or more embodiments, the first dispersion medium and the seconddispersion medium are water.

In one or more embodiments, the first dispersion medium and the seconddispersion medium are removed by evaporation.

The present disclosure provides a method for maintaining growth andstemness of cells, comprising culturing the cells on the membrane asdescribed in the present disclosure.

In one or more embodiments, the cells are selected from the groupconsisting of induced pluripotent stem cells, embryonic stem cells andadult stem cells.

In one or more embodiments, the method for maintaining growth andstemness of cells comprises

(a) inoculating the cells onto the membrane according to the presentdisclosure, and

(b) maintaining the cells in a first medium.

In one or more embodiments, the cells are selected from the groupconsisting of induced pluripotent stem cells, embryonic stem cells andadult stem cells.

EXAMPLES

The embodiments of the present disclosure will be described in detailbelow with reference to examples, but those skilled in the art willunderstand that the following examples are only intended to illustratethe present disclosure, and should not be construed as limiting thescope of the present disclosure. Examples are carried out according tothe conventional conditions or the conditions recommended by themanufacturer, if specific conditions are not described. All reagents orinstruments used, whose manufacturers are not indicated, arecommercially available conventional products.

Example 1. Preparation of BCC Membrane

The multifunctional particle membrane of this example is composed of twokinds of different particles, wherein the large particles are inorganicsilica particles, and the small particles are organic polymer particles.2SiPM (also referred to as 2PM herein) consists of 2 μm silicamicrospheres (SiO₂) and 0.1 μm poly(methyl methacrylate) (PMMA)nanoparticles, 5SiPM (also referred to as 5PM herein) consists of 5 μmSiO₂ microspheres and 0.2 μm PMMA nanoparticles, 2SiPS (also referred toas 2PS herein) consists of 2 μm SiO₂ microspheres and 0.2 μm polystyrene(PS) nanoparticles, and 5SiPS (also referred to as 5PS herein) consistsof 5 μm SiO₂ microspheres and 0.4 μm PS nanoparticles.

The preparation process comprises the following steps. Two kinds ofparticles of different sizes were separately dispersed in water toobtain colloidal solutions, and the two colloidal solutions are mixedwell, wherein the mixing volumes of the two kinds of particles arecalculated in such a manner that the particles can form a single-layermembrane on a surface of a coverslip. Then, the mixture was addeddropwise to a coverslip (Solarbio, YA0352), water was evaporated toself-assemble the particles on the surface of the coverslip to form amulti-stage structure (see FIG. 2), and finally the surface of themulti-stage structure was stabilized by heating. Sterilization isperformed by UV irradiation before use.

The surface structure of the four materials was characterized byscanning electron microscopy, with detailed structure shown in FIG. 3.It can be seen that the large particle component (silica) is spread onthe surface of the coverslip in a single layer and partially embedded inthe small particle component (organic polymer). Thus, a surface composedof a single-layer dispersed micrometer-sized (2 or 5 μm) large particlesand nano-sized (100 or 400 nm) small particles is formed. The sizes andcombinations of the two kinds of particles are advantageous for celldifferentiation.

Example 2. hiPSC Cell Culture

Four BCCs membranes and a control coverslip were coated with Matrigel(Corning, 354277). The hiPSC cell line NC5 (Help Stem Cell Innovations,NC2001) was inoculated onto the coated surface and expanded, and theseeding density was 1×10⁶/10 cm². The cells were maintained in a mTeSRmedium (Stemcell, 05850) with 5% CO₂ and at 37° C.

Cardiac Differentiation of hiPSC

Directed cardiac differentiation of hiPSCs involves the following steps.When hiPSCs reached 80% confluence, cardiac differentiation of hiPSCswas started, and the medium was changed to RPMI1640 (Gibco, 1744361)with B-27(Gibco, A1895601). For the early stages of differentiation, thecells were exposed to the GSK313 inhibitor CHIR 99021 (6 μM, Selleck,S2924) followed by the Wnt antagonist IWR-1 (5 μM, Sigma-Aldrich,10161). Contracting cells were noted from day 8 and were fed everyalternate day with RPMI1640 medium (Gibco, 17504-044) supplemented withthe B-27 supplement. During day 15-20, the medium was changed to apurification medium, which consists of a glucose-free Dulbecco'smodified Eagle's medium (Gibco, 11966025) supplemented with 4 mM lacticacid and sterile 1M Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES). After 30 days of in vitro differentiation, the cells weretrypsinized and replated on gelatin-coated coverslips (Solarbio, YA0352)for further experiments.

See FIG. 4 for the process of cell culture and differentiation ofhiPSCs.

It should be noted that the steps of trypsinizing the differentiatedhiPSC cells and replating the same on gelatin-coated coverslips in thisexample serve for the subsequent analysis of hiPSC-CM cells. In theproduction process of the hiPSC-CM cells with a purpose of preparation,the enzymolysis step is not required, instead, cell spheroids can beseparated from the BCC substrate directly by flushing with, for example,buffer or medium. That is, the method in the present example does notrequire the steps of first suspension-culturing cells into EBs and theninoculating the same onto a substrate in the 2D induced differentiationmethod in the prior art.

Unlike the 3D induced differentiation method in the prior art, themethod in this example does not require the steps ofsuspension-culturing the hiPSCs in advance to generate EBs and thenimplanting the same on the substrate, but the cell culture anddifferentiation steps of the hiPSCs are always performed on the surfaceof the BCC.

Example 3. Analytical Method

Immunofluorescence Staining

Cells were fixed with 4% paraformaldehyde (PFA) in Dulbecco'sphosphate-buffered saline for 20 min at room temperature, andpermeabilized with 0.1% Triton-X 100 for 10 min. The cells were thenincubated with the following primary antibodies overnight at 4° C.:rabbit anti-Oct4 (CST, 2750S) and mouse anti-SSEA-4 (R&D, MAB1435) forpluripotency staining of hiPSCs; rabbit anti-a-tubulin (abeam, ab18251)and mouse anti-α-actinin (Sigma, A7811) for structural staining ofhiPSC-CMs; and rabbit anti-N-Cadherin (abeam, ab76057) and mouseanti-E-Cadherin (abeam, ab1416) for cell adhesion staining. Thesecondary antibodies were donkey antirabbit IgG (Alexa Fluor 488, abeam,ab150073) and goat antimouse IgG (Alexa Fluor 555, abeam, ab150118).Nuclei were visualized with DAPI (Beyotime, C1006). Images were capturedusing a Zeiss fluorescence microscope (Zeiss, Axio Vert A1).Measurements of the cardiomyocyte size were performed with ImageJsoftware (National Institutes of Health, 1.8.0_77) [Wang, J., et al.Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived fromHuman Induced Pluripotent Stem Cells. ACS Appl. Mater. Interfaces 2017,9, 25929-25940.]. The aspect ratio was defined as long axes/short axes.The circularity index was defined as 4π area/perimeter².

Flow Cytometry

Signal cardiomyocytes were obtained with trypsin and fixed in 4% PFA for20 min. To analysis intracellular proteins, the cells were permeabilizedwith 0.1% Triton-X 100 for 10 min. The following primary antibodies wereapplied: rat anti-SSEA3 (Alexa Fluor 488, R&D, FAB1434G), rabbitanti-cardiac troponin I (Alexa Fluor 488, abeam, ab196384), rabbitanti-MYL7 (PE, Miltenyi, 130-117-546), and mouse anti-MYL2 (Alexa Fluor488, Novus, NBP1-30249G). The stained cells were counted using BD FACSCalibur. Data analysis was performed using FlowJo software [Liang, P.,et al. Drug screening using a library of human induced pluripotent stemcell-derived cardiomyocytes reveals disease-specific patterns ofcardiotoxicity. Circulation 2013, 127, 1677-1691; Nunes, S., et al aplatform for maturation of human pluripotent stem cell-derivedcardiomyocytes. Nat. Methods 2013, 10, 781-787.].

Patch Clamp Assay

Whole-cell patch clamp was used to record the APs (action potentials) onthe Axopatch 200B amplifier (Axon), comprising the following steps andconditions. AP was examined using the following intracellular solutions:120 mM K-aspartate, 25 mM KCl, 5 mM Mg₂ATP, 1.8 mM CaCl₂, 5 mM HEPES, 10mM ethylene glycolbis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and 10 mM glucose (pH 7.3). The composition of external Tyrode'ssolution was as follows: 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.3). For the specificprocedure of the patch clamp method, please refer to Nunes, S. S.;Miklas, J. W.; Liu, J.; Aschar-Sobbi, R.; Xiao, Y.; Zhang, B.; Jiang,J.; Masse, S.; Gagliardi, M.; Hsieh, A.; Thavandiran, N.; Laflamme, M.A.; Nanthakumar, K.; Gross, G. J.; Backx, P. H.; Keller, G.; Radisic, M.Biowire: a platform for maturation of humanpluripotent stem cell-derivedcardiomyocytes. Nat. Methods 2013, 10, 781-787.

Quantitative RT-PCR

RNA was isolated with Trizol (Invitrogen) following manufacturer'sinstructions. cDNA was produced using the iScript cDNA Synthesis Kit(170-8891, Bio-Rad). Real-time PCR was conducted using the SYBR greenPCR kit (170-8882AP, Bio-Rad) and performed on a 7900HT Real-Time PCRSystem (Life Technologies). All PCRs were in quadruplicate andnormalized to β-actin or GAPDH which were considered as the housekeepinggenes. Study procedures were approved by the Bioethics Committee of theFirst Affiliated Hospital of Nanjing Medical University (2014-SR-090).Human adult heart tissues collected during the surgical procedures weresnap-frozen in liquid nitrogen, followed by protein analysis andquantitative real-time PCR (qRT-PCR).

Western Blot

The primary antibodies used in the present study were as follows: GAPDH(1:1000, Cell Signaling, 2118S), N-cadherin (1:1000, abcam, ab76057),E-cadherin (1:1000, abcam, ab1416), cardiac troponin I (cTnI; 1:1000,abcam, ab47003), and ssTnI (1:1000, Abcam, ab8293). Secondary antibodiesemployed in this assay included the anti-rabbit IgG antibody (1:5000,Cell Signaling, 7074P2) or anti-mouse IgG antibody (1:5000, CellSignaling, 7076). For the detailed procedure of Western blottingdetection of N-Cadherin, please refer to Cui, C.; Geng, L.; Shi, J.;Zhu, Y.; Yang, G.; Wang, Z.; Wang, J.; Chen, M. Structural andelectrophysiological dysfunctions due to increased endoplasmic reticulumstress in a long-term pacing model using human induced pluripotent stemcell-derived ventricular cardiomyocytes. Stem Cell Res. Ther. 2017, 8,109.

Statistical Method

Statistical analysis was performed using SPSS Statistics 19.0. Normallydistributed data were shown as the means (estimated by one-way analysisof variance (ANOVA))±standard deviation (SD) followed by the Dunnettest. Skew distributed data were expressed as the median (interquartilerange), estimated by the Nemenyi test. A p-value less than 0.05 wasconsidered statistically significant.

Example 4. Effect of BCCs on the Culture of hiPSC Cells

Two days after the start of the cell differentiation, hiPSCs grew intomonolayers on 2PS, 5PS, and control surfaces. However, hiPSCs formedstereoscopic clusters on 2PM and 5PM surfaces (see FIG. 5A). hiPSCclusters were slightly larger with denser cells on the 5PM surface thanon the 2PM surface. It is reported that the morphology of hiPSCs is animportant factor of its pluripotency. The pluripotency of hiPSCs wasconfirmed by qRT-PCR on OCT4, NANOG and SOX2. The results showed thathigher gene expression was found in the PM group, particularly on the5PM surface, compared to the PS group and the control group (n=4, FIG.5B).

Example 5. Effect of BCCs on Cardiac Differentiation of hiPSCs

Cardiac differentiation of hiPSCs was induced with small molecules (FIG.4). Eight days after the start of the differentiation, spontaneousbeating cardiomyocytes were observed on all surfaces. The morphology ofhiPSC-CMs was still 2D-like cell sheets on the control and PS groups,while these formed 3D-like spheroids in PM groups.

It has been reported that MLC2v-positive result means ventricular-likecardiomyocytes, while MLC2v-positive/MLC2a-negative result means matureventricular-like cardiomyocytes [Minami, I., et al. A small moleculethat promotes cardiac differentiation of human pluripotent stem cellsunder defined, cytokine- and xeno-free conditions. Cell Rep. 2012, 2,1448-1460; Tiburcy, M., et al. Defined Engineered Human Myocardium WithAdvanced Maturation for Applications in Heart Failure Modeling andRepair. Circulation 2017, 135, 1832-1847.]. In chamber-specificanalysis, a similar proportion of MLC2v-positive hiPSC-CMs (about 95%)was found on all surfaces, suggesting that the majority of hiPSC-CMswere ventricular-like cardiomyocytes. However, the ratio of matureventricular cardiomyocytes, defined as MLC2v⁺/MLC2a⁻ cells, was higheron PM groups (about 80%) than on PS groups and the control (<60%),suggesting that PM surfaces promoted the pronounced maturation ofhiPSC-CMs (FIG. 6). hiPSCs on PM substrates formed 3D-like spheroidswhich had stronger cell-cell contacts and higher pluripotency comparedto those monolayer hiPSCs on PS and control surfaces. This phenomenon inturn generates functional and mature hiPSC-CMs compared to controls.

Example 6. PM Substrates Improved Cardiac Structural Maturation ofhiPSC-CMs

Previous studies suggest that adult cardiomyocytes exhibit an elongatedshape with organized myofibrils [Gerdes, A. M.; et al. Structuralremodeling of cardiac myocytes in patients with ischemic cardiomyopathy.Circulation 1992, 86, 426-430.]. 30 days after the differentiation,hiPSC-CMs on different surfaces were trypsinized into single cells andreplated on gelatin-coated plates. In detail, single hiPSC-CM from PMgroups was more elongated, while those from PS groups and the controlwere less elongated (FIG. 7A). The spatial organization of myofibrilswas highly ordered and clear on BCCs but not on controls. α-Actinin isparallel and anisotropic when cells were elongated, which was observedin PM-derived hiPSC-CMs.

Quantitative analyses of cell morphology including the aspect ratio,circularity, spreading area, and parameter of cells showed that theaspect ratio of 5PM-derived hiPSC-CMs was significantly higher and thecircularity of PM-derived hiPSC-CMs was significantly lower compared tothe controls (n=40-44, FIG. 7B and FIG. 7C).

Another mature indicator, myofibrils or the length of sarcomeres, isdirectly related to the contraction force of cardiomyocytes [Kentish, J.C., et al. Comparison between the sarcomere length-force relations ofintact and skinned trabeculae from rat right ventricle. Influence ofcalcium concentrations on these relations. Circ. Res. 1986, 58,755-768.]. The length of sarcomeres was significantly longer onPM-derived hiPSC-CMs compared to other surfaces (FIG. 7D).

Taken together, these results indicate that PM surfaces promoted thestructural maturation of hiPSC-CMs.

Example 7. PM Substrates Improved Maturation of ElectrophysiologicalProperties of hiPSC-CMs

To verify the electrophysiological properties, the AP was analyzed usingthe patch-clamp method [Nunes, S. S., et al. Biowire: a platform formaturation of human pluripotent stem cell-derived cardiomyocytes. Nat.Methods 2013, 10, 781-787.]. AP duration at 50% and 90% repolarization(APD50 and PAD90) was measured by the patch-clamp method. The resultsshowed that PM surfaces prolonged APD50 and APD90 (n=10, FIG. 9B andFIG. 9C). Moreover, the ratio of APD50/APD90 was greater than 0.8 on PMsurfaces, while that on the PS surfaces and control was about 0.7 (FIG.9D). The result again suggested that PM substrates promoted hiPSC-CMstoward more mature ventricular cardiomyocytes.

qRT-PCR was performed to test the expressions of essential cardiac genesin hiPSC-CMs 30 days after the differentiation on BCCs including cardiacstructural component genes (ACTC1 and TNNT2), Ca²⁺ transient-relatedproteins (RYR2 and SERCA2a), major cardiac ion-channel genes (SCN5a,KCNJ2, and CACNA1c), cell membrane surface genes (ITGB1 responsible forinformation transfer between cells and extracellular matrix, and GJA1responsible for cell-to-cell gap junction intracellular communication toregulate cell death), and myofibrillar isoforms (MYH6 and MYH7).Consistent with functional experiments, PM substrates, especially 5PM,significantly enhanced the expressions of these critical cardiac genes,which expressions were higher than the expressions of these genes inadult myocardium (n=4, FIG. 8). Notably, myosin heavy chain (MHC)isozymic transitions from MHC-α to MHC-β were known as cardiacdevelopment/maturation markers [Yoshida, S., et al. Maturation of HumanInduced Pluripotent Stem Cell-Derived Cardiomyocytes by Soluble Factorsfrom Human Mesenchymal Stem Cells. Mol. Ther. 2018, 26, 2681-2695.]. Inthe present example, the 5PM surface significantly increased the RNAexpressions of MHC-β (MYH7), whereas the expressions of MHC-α (MYH6) didnot differ among five surfaces. Altogether, 5PM surfaces promotematuration of hiPSC-CMs compared to PS surfaces and the 2D control.

Example 8. 5PM Substrate Promoted the N-Cadherin Expression DuringCardiogenesis

The BCC-induced higher cell-cell contact is crucial in cardiacdifferentiation of hiPSCs. This effect was verified by analyzingcadherins, cell adhesion molecules. During cardiogenesis, the expressionof E-cadherin is gradually suppressed with a gradual increase ofN-cadherin expression. This process is known as cadherin switching, ahallmark of epithelial-to-mesenchymal transition (EMT) [Karimzadeh, F.,et al. Calreticulin Is Required for TGF-β-InducedEpithelial-to-Mesenchymal Transition during Cardiogenesis in MouseEmbryonic Stem Cells. Stem Cell Rep. 2017, 8, 1299-1311; Brade, T., etal. Embryonic heart progenitors and cardiogenesis. Cold Spring HarborPerspect. Med. 2013, 3, a013847.]. It has also been reported that thepresence of N-cadherin at the cell surface of cardiomyocytes isnecessary for the contraction [Zhang, J., et al. Extracellular MatrixPromotes Highly Efficient Cardiac Differentiation of Human PluripotentStem Cells. Circ. Res. 2012, 111, 1125-1136; Luo, Y., et al. Rescuingthe N-cadherin knockout by cardiac-specific expression of N- orE-cadherin. Development 2001, 128, 459-469; Bagatto, B., et al.Lingua::EN::Titlecase. BMC Dev. Biol. 2006, 6, 23.]. Herein, hiPSC-CMson PM substrates formed 3D-like spheroids, indicating an increase ofcell-cell contacts.

Immunostaining showed that the expression of N-cadherin was stronger onPM surfaces than on other surfaces, while E-cadherin has similar lowintensity across surfaces after 8 days (FIG. 10A).

Owing to the topography of BCC materials, it is not easy to obtain theclear picture from a specific stack. Hence, further western blotanalysis was carried out. N-Cadherin expression was detected by Westernblot. Consistently, it demonstrated that the 5PM surface significantlyincreased the expression of N-cadherin, while the expressions ofE-cadherin showed no differences among all the groups (n=3, FIG. 10B andFIG. 10C).

These results indicated that PM substrates provided optimizedbiophysical stimulation through cell-surface and cell-cell interactionsto hiPSCs which maintained the pluripotency of hiPSCs during expansionand facilitated cardiac differentiation of hiPSCs induced by smallmolecules.

In summary, BCCs are versatile platforms that have multidimensionalstructures and heterogeneous chemistries. These are important modulatorsin the cell microenvironment. BCCs with different combinations can befabricated in a short period of time which can be used to screen thecell-substrate interaction. Using this high-throughput screeningplatform, the BCCs optimized in modulation of the hiPSC behavior can beobtained. hiPSC expansion and cardiac differentiation can be performedon BCCs using the one-step method without EB formation. Moreimportantly, mature and functional hiPSC-CMs can be generated onoptimized BCCs, that is, 5PM surfaces. The mechanism was answered thatsurface properties of PM groups altered cell morphology and facilitatedcell-cell contacts and cadherin switching during cardiacdifferentiation. Improvement of the N-cadherin expression enhanced EMTthat benefited the differentiation of hiPSCs into globally maturecardiac cells. The method of the present disclosure provided a simpleand efficient way to produce mature hiPSC-CMs.

p1) The method for culture and differentiation of cells the presentdisclosure using the membrane of the present disclosure is a one-stepmethod. That is, the culture and differentiation of pluripotent stemcells, such as hiPSCs, are carried out on a multi-scale particlemembrane. Compared with the existing 3D culture technology which is atwo-step method including forming EBs first, and then transferring tothe 3D scaffold for differentiation, the method of the presentdisclosure is time and effort saving, and reduces the chance to damageand contaminate cells. In contrast to the existing 2D culturetechniques, in which trypsin digestion is required to separate culturedor differentiated cells from substrate, the method of the presentdisclosure can separate cultured or differentiated cells from themembrane using a simple means such as flushing, thus simplifying theprocess and reducing damages to cells.

2) The membrane and cell culture method of the present disclosure mayallow stem cells, such as hiPSCs, to grow in the form of 3D-likespheroids, maintaining their pluripotency and stemness.

3) Using the membrane and culture method of the present disclosure, moremature differentiated stem cells, such as hiPSC-CMs, can be obtained ina same period of time compared with the traditional methods.

What is claimed is:
 1. A membrane for culture and differentiation ofcells, comprising: (A) a base portion; and (B) a protrusion arraycomposed of a plurality of protrusions, the plurality of protrusionsbeing substantially uniformly distributed on the base portion, and theplurality of protrusions having a size on the order of micrometers. 2.The membrane according to claim 1, wherein a distance between adjacentprotrusions is on the order of micrometers.
 3. The membrane according toclaim 1, wherein the membrane is a colloidal crystal membrane.
 4. Themembrane according to claim 1, wherein the membrane is composed ofparticles of different particle sizes of two or more kinds, and the twoor more kinds of particles of different particle sizes comprise atleast: particles of a first kind, serving as the protrusions and havingan average particle size of 1 μm to 50 μm, and particles of a secondkind, serving as the base portion and having an average particle sizeless than or equal to ½ of the average particle size of the first kindof particles.
 5. The membrane according to claim 1, wherein the membraneis composed of particles of different particle sizes of two kinds,wherein in the particles of different particle sizes of the two kinds,particles of a first kind have an average particle size of 1 μm to 50μm, and particles of a second kind have an average particle size of10-900 nm.
 6. The membrane according to claim 5, wherein the particlesof the first kind are made of an inorganic compound.
 7. The membraneaccording to claim 5, wherein the particles of the second kind are madeof an organic polymer.
 8. The membrane according to claim 5, wherein theparticles of the first kind are made of one or more materials selectedfrom the group consisting of silica, titania, zinc oxide, chemicallymodified silica, chemically modified titanium dioxide, chemicallymodified zinc oxide, and any combination thereof.
 9. The membraneaccording to claim 5, wherein the particles of the second kind are madeof one or more materials selected from the group consisting ofpolystyrene, acrylic polymers, chitosan, poly(lactic-co-glycolic acid),polylactic acid, polycaprolactone, gelatin and any combination thereof.10. The membrane according to claim 9, wherein the acrylic polymers areselected from the group consisting of poly(meth)acrylic acids,poly(meth)acrylates and any combination thereof.
 11. The membraneaccording to claim 10, wherein the poly(meth)acrylates are selected fromthe group consisting of poly(meth)acrylic acid C₁-C₂₀ alkyl esters. 12.The membrane according to claim 10, wherein the poly(meth)acrylates areselected from the group consisting of poly(methyl acrylate), poly(methylmethacrylate), poly(ethyl acrylate), poly(ethyl methacrylate),poly(propyl acrylate), poly(propyl methacrylate), poly(butyl acrylate),poly(butyl methacrylate), poly(pentyl acrylate), poly(pentylmethacrylate), poly(hexyl acrylate), poly(hexyl methacrylate) and anycombination thereof.
 13. The membrane according to claim 5, wherein theparticles of the first kind are made of silica and the particles of thesecond kind are made of poly(methyl methacrylate).
 14. The membraneaccording to claim 5, wherein the particles of the first kind are inform of a single layer of particles, and a ratio of the particles of thefirst kind and the particles of the second kind is set such that theparticles of the first kind are distributed in the particles of thesecond kind in a partially embedded manner to form a colloidal crystal.15. A method for culture and/or differentiation of cells, comprisingculturing and/or differentiating the cells on the membrane according toclaim
 1. 16. The method according to claim 15, wherein the cells areselected from the group consisting of induced pluripotent stem cells,embryonic stem cells and adult stem cells.
 17. The method according toclaim 15, wherein the cells are selected from the group consisting ofbone marrow mesenchymal stem cells, hematopoietic stem cells, neuralstem cells, peripheral blood stem cells, adipose stem cells, placentalstem cells, placental sub-totipotent stem cells, and amniotic stemcells.
 18. The method according to claim 15, wherein the cells are humaninduced pluripotent stem cells, and by the method, the human inducedpluripotent stem cells are differentiated in a directed differentiationmanner into human induced pluripotent stem cell-derived cardiomyocytes.19. The method according to claim 15, further comprising separating thecells from the membrane by liquid flushing or suction after thedifferentiation.
 20. A method for maintaining growth and stemness ofcells, comprising culturing the cells on the membrane according to claim1, wherein the cells are selected from the group consisting of inducedpluripotent stem cells, embryonic stem cells and adult stem cells.